Functional surface expression of immunoglobulin cleavage systems in a candidate Mycoplasma vaccine chassis

The Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system is a candidate ‘virulence factor present in multiple pathogenic species of the Mollicutes, including the fast-growing species Mycoplasma feriruminatoris. The MIB-MIP system cleaves the heavy chain of host immunoglobulins, hence affecting antigen-antibody interactions and potentially facilitating immune evasion. In this work, using -omics technologies and 5’RACE, we show that the four copies of the M. feriruminatoris MIB-MIP system have different expression levels and are transcribed as operons controlled by four different promoters. Individual MIB-MIP gene pairs of M. feriruminatoris and other Mollicutes were introduced in an engineered M. feriruminatoris strain devoid of MIB-MIP genes and were tested for their functionality using newly developed oriC-based plasmids. The two proteins are functionally expressed at the surface of M. feriruminatoris, which confirms the possibility to display large membrane-associated proteins in this bacterium. However, functional expression of heterologous MIB-MIP systems introduced in this engineered strain from phylogenetically distant porcine Mollicutes like Mesomycoplasma hyorhinis or Mesomycoplasma hyopneumoniae could not be achieved. Finally, since M. feriruminatoris is a candidate for biomedical applications such as drug delivery, we confirmed its safety in vivo in domestic goats, which are the closest livestock relatives to its native host the Alpine ibex.

Bacteria of the class Mollicutes are characterized by the absence of a cell wall and numerous enzymatic pathways that were lost by reductive evolution from Gram-positive ancestors.As a result, Mollicutes have a pleomorphic cell shape and live a parasitic lifestyle to scavenge nutrients from their host.A number of Mollicutes infecting plants or animals (including humans) are pathogenic, such as the well-known pathogens Candidatus Phytoplasma asteris 1 , members of the "Mycoplasma mycoides cluster" 2 and Mycoplasmoides (Mycoplasma) pneumoniae and Mycoplasmoides (Mycoplasma) genitalium 3,4 , respectively.Knowledge of their virulence traits is still scarce due to the fastidious nature of these organisms and the historical lack of genetic tools to modify their genomes.Recently, the candidate virulence factor Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system has been characterized in various Mollicutes species [5][6][7][8] .This system consists of at least two surface located proteins that bind and cleave the variable region of the heavy chain (V H ) of IgGs and has been shown to be active in vivo in goats infected with Mycoplasma mycoides subsp.capri (Mmc) 9 .The structure of the two proteins in complex with an antibody was solved using cryo-electron microscopy.The two proteins bind to the Fab fragment in a "hug of death" mechanism, which is thought to interfere with antibodyantigen interactions 10 .
Mycoplasma feriruminatoris (Mferi) is a close relative of the Mollicutes belonging to the "M.mycoides cluster" and has been isolated from Alpine ibex and Rocky Mountain goats 11,12 .This species is characterized by its short doubling time compared to the slow growth typically observed in many other Mollicutes.The genome of Mferi has been recently adapted to synthetic genomics techniques 13 , including genome editing in Saccharomyces cerevisiae and genome transplantation 14 .All these features together with the simplistic nature of Mollicutes, absence of a cell wall and different genetic code have turned Mferi and other Mollicutes as promising candidates to serve as a workhorse for industrial applications 15 , such as in vivo vaccine or drug delivery vessel 13,16,17 .
In this work we analyzed in depth the operons expressing the four MIB-MIP gene copies present in Mferi by using -omics technologies and cDNA amplification.We also developed oriC-based plasmids to allow rapid introduction of genes and in cellulo expression of homologous and heterologous DNA at a high turnaround time.Using these tools, we could express functional MIB-MIP gene tandems individually to test their activity and we also attempted expression of heterologous MIB-MIP systems from other Mollicutes species, proving that Mferi is a valuable bacterial platform for functional genomics studies of this and other species.Finally, we also tested the pathogenicity of Mferi in domestic goats in vivo and demonstrated its safety, paving the way for future industrial applications.

Materials and methods
All methods employed in this work that required use of commercial kits were performed following the manufacturer's instructions, unless stated otherwise.
All primers used in this work for the different applications can be found listed in Supplementary Table S1.

Strains used and culture conditions
Escherichia coli Stellar cells (Clontech) or NEB 5-alpha (New England Biolabs) were used for all constructions of different oriC-plasmids and subsequent plasmid preparations.All E. coli strains were cultured in Luria Bertani (LB) medium at 37 °C and shaking at 220 rpm or on LB agar plates supplemented with 100 µg mL −1 ampicillin when necessary.Transformation of E. coli strains was achieved by using a heat-shock standard protocol 18 .
S. cerevisiae strain W303a was used to modify and propagate the Mferi genome.S. cerevisiae was cultured in Yeast Peptone Dextrose Adenine or Synthetic Defined broth (Formedium) depleted for tryptophan, uracil and/ or histidine depending on the auxotrophic marker in use.S. cerevisiae strains were cultured at 30 °C and 220 rpm.
Mferi IVB14/OD_0535 15 , Mesomycoplasma (Mycoplasma) hyorhinis JF5820 19 , and Mesomycoplasma (Mycoplasma) hyopneumoniae Ue273 used in this study were isolated from diagnostic material at the Institute of Veterinary Bacteriology in Bern.M. hyopneumoniae Ue273 was isolated from bronchial tissue of a Swiss wild boar.Mferi and Mmc strains used for -omics and in vitro studies were grown at 37 °C with 5% CO 2 in SP5 medium 20 or modified Hayflick agar plates 21 supplemented with 15 µg mL −1 tetracycline or 16 µg mL −1 puromycin when necessary.Presence of oriCplasmids in liquid cultures was maintained by puromycin (8 µg mL −1 ).M. hyorhinis and M. hyopneumoniae were grown in Friis medium 22 at 37 °C.Mycoplasma capricolum subsp.capricolum ΔRE (Mcap ΔRE) was used as a recipient for genome transplantation from yeast 23 (see "Genome transplantation" section below).For the experimental infection M. capricolum subsp.capripneumoniae (Mccp) ILRI181 and Mferi G5847 T were grown at 37 °C with 5% CO 2 in Mycoplasma Experience Liquid Medium (Mycoplasma Experience), aliquoted and stored at −80 °C until used.

Phylogenetic analysis
The phylogenetic analysis based on the 16S rRNA gene sequences of the Mollicutes covered overall n = 7 genera, encompassing n = 27 species and n = 36 strains.The tree was built in BioNumerics v8.1 using Jukes-Cantor correction and the Neighbour Joining method.Clostridium innocuum was used as outgroup.
For the phylogenetic analysis of the MIB-MIP system, the translated amino acid sequences were used using the genomes described above (Supplementary Data 1).Orphan copies of either the MIB or MIP were not included in the analysis.MIB and MIP protein sequences were first aligned separately with MAFFT v7.5 24 , then the alignments were concatenated with a custom R script.Phylogenetic reconstruction of the unrooted tree was performed with IQ-TREE 2.0.3 25,26 for Linux, running with phyml parameter and 1000 replicates for both ultrafast bootstrap 27 and likelihood ratio test.ModelFinder 28 gave the LG+F+I+G4 model as best-fit.The tree was then plotted and manually edited in FigTree (v1.4.4).

Genomic DNA extraction and Next Generation Sequencing
Genomic DNA (gDNA) from Mollicutes was extracted from 20 mL cultures using the Promega Wizard Genomic DNA purification kit.The quality and quantity of the gDNA was assessed on agarose gels and using the Qubit fluorometer (Invitrogen).Subsequently, gDNA was sequenced in the Pac-Bio sequencing platform at the Lausanne Genomic Technologies Facility at the Center for Integrative Genomics (University of Lausanne), as described elsewhere 29 .High molecular weight DNA was sheared with Megaruptor (Diagenode, Denville, NJ, USA) to obtain 10 kb fragments.After shearing the DNA size distribution was checked on a Fragment Analyzer (Advanced Analytical Technologies, Ames, IA, USA).500 ng of each DNA was used to prepare a SMRTbell library with the PacBio SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences).The resulting libraries were pooled with other libraries processed the same.The pool was size selected with Ampure PacBio beads to eliminate fragments <5 kb.It was sequenced with v3.2/v2.0chemistry and diffusion loading on a PacBio Sequel II instrument (Pacific Biosciences) at 900 min movie length and pre-extension time of 120 min using one SMRT cell 8M.Demultiplexing, microbial assemblies and m6A-m4C base modification detection were performed using smrtlink v11.1.Genomes were assembled from PacBio reads with Unicycler v0.4 30 , smrtlink v11 or Flye v2.8 31 .Circularized genomes were polished with three rounds with the Arrow software [single-molecule real-time (SMRT) Link version 8 package].Genomes were rotated to the first nucleotide of the start codon of the dnaA gene, and annotated using Prokka, version 1.13 31 .M. hyopneumoniae Ue273, Mesomycoplasma (Mycoplasma) ovipneumoniae 14KM848 and Mferi ΔMIB-MIP sequences are deposited as BioProject PRJNA1062711.
Transcriptomic analysis and 5' Rapid Amplification of cDNA Ends RNA extraction for transcriptomics analysis was carried out as previously described 29 .Briefly, RNA from three biological replicates of Mferi was extracted from 5 mL liquid cultures using the Zymo Research Quick-RNA Fungal/Bacterial Miniprep kit.RNA quality was assessed at the Lausanne sequencing platform on a Fragment Analyzer (Agilent Technologies).Libraries were prepared using the Illumina TruSeq Stranded mRNA reagents (Illumina), excluding the polyA selection step and using a unique dual indexing strategy.Ribosomal rRNA depletion was carried out with QIAseq FastSelect-5S/16S/23S kit (Qiagen).Libraries were quantified by Qubit (Life Technologies) and their quality was assessed on a Fragment Analyzer (Agilent Technologies).Cluster generation was performed with 1.92 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents and sequenced on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 2 × 150 cycles (paired end).Sequencing data were demultiplexed using the bcl2fastq2 Conversion Software v. 2.20 (Illumina).Reads were first trimmed with fastp (0.19.5) and aligned against the reference genome (LR739236.1)with bwa mem (0.7.13).The mapped reads were filtered for quality, duplication and pairing with picard-tools (2.9.0) (http://broadinstitute.github.io/picard)and samtools (1.10) 32 .RNAseq coverage analysis was performed using defaults parameters from geneBody_coverage and FPKM_count from the RSeQC (v5) package 33 .Then resulting coverage and histograms plots were done in R.
5' Rapid Amplification of cDNA Ends (5'RACE) was performed using the 5'/3'RACE kit (Roche), except for the PCR amplification steps, in which dA-tailed cDNA was amplified using Q5 polymerase (New England Biolabs) instead.The PCR conditions were the following: initial denaturation at 98 °C for 2 min; 10 cycles of 15 s at 98 °C, 30 s at 54 °C and 40 s at 72 °C; 10 cycles of 15 s at 98 °C, 30 s at 54 °C and 90 s at 72 °C; 15 cycles of 15 s at 98 °C, 30 s at 54 °C and 180 s at 72 °C; and a final extension of 7 min at 72 °C.The resulting PCR products were diluted 20x in distilled water and 1 µL was used for the subsequent nested PCR, using the same PCR protocol except that the annealing temperature in all cycles was increased to 56 °C.Final PCR products were purified using High Pure PCR product Purification Kit (Roche) and visualized in a 2% agarose gel.Purified PCR products were Sanger sequenced using the gene specific primers (Microsynth) and also assembled into pUC57mini vectors (Genscript) using the NEBuilder Hi-Fi DNA Assembly kit (NEB) prior transformation into E. coli DH5α.Several clones resulting from transformation were further screened by PCR and Sanger sequencing (Microsynth).

Proteomics analyses
Proteomics analyses were performed as previously described 29 .Briefly, the same three biological replicates of mycoplasmas used for transcriptomics were harvested by centrifugation at 4000 × g at 4 °C for 15 min.Pellets were washed three times in ice-cold PBS and stored at −80 °C until further use.Cell pellets were reconstituted in 500 µL 8 M urea, 100 mM Tris/HCl pH 8, protein concentration determined with Pierce BCA Protein Assay Kit (ThermoScientific), and cysteines alkylated with 50 mM iodoacetamide for 30 min in the dark after reduction with 10 mM DTT for 30 min at 37 °C34 .Proteins were precipitated with five volumes of cold acetone over night at −20 °C.Proteins were dissolved in 8 M urea, 50 mM Tris/HCl pH 8 at a concentration of 1 mg mL −1 .An aliquot of 10 µL was further processed by lowering the urea concentration to 1.6 M with 20 mM Tris/HCl pH 8 containing 2 mM CaCl 2 , followed by digestion with 200 ng trypsin over night at room temperature.After acidification with 1% trifluoroacetic acid end concentration, aliquots of 5 µL (500 ng protein digest) were analyzed by nano-liquid reversed phase chromatography coupled to tandem mass spectrometry on an Orbitrap Fusion LUMOS mass spectrometer that was coupled with a Dionex Ultimate 3000 nano-UPLC system (ThermoFischer Scientific).A standard data-dependent acquisition method as described elsewhere 35 was used with a homemade AcquityTM CSH C18 separation column (1.7 µm, 130 Å, 75 µm × 20 cm) at a flow rate of 250 nL min −1 .
Mass spectrometry-derived proteomic data were analyzed against LR739236.1 Genbank file with concatenated reverse sequence decoys by Transproteomic pipeline (TPP) tools 36 .The five database search engines Comet 37 , Xtandem 38 , MSFragger 39 , MS-GF+ 40 , and MyriMatch 41 were used and each search was followed by the application of the PeptideProphet tool 42 .The iProphet 43 software was subsequently used to summarize the search results, which were filtered at the false discovery rate of 0.01; furthermore, identifications were exclusively accepted if at least three of the search engines agreed on the identification.Protein inference was performed using ProteinProphet.For those protein groups accepted by a false discovery rate filter of 0.01, a Normalized Spectral Abundance Factor (NSAF) 44 was calculated based on the peptide to spectrum match count.Shared peptides were considered by a method published elsewhere 45 .The mass spectrometry proteomics data have been deposited to the Proteo-meXchange Consortium via the PRIDE 46 partner repository with the dataset identifier PXD053286.
CReasPy-Cloning of M. feriruminatoris genome A mutant strain of Mferi IVB14/OD_0535 without the four MIB-MIB gene pairs (ΔMIB-MIP) was generated using the CReasPy-Cloning method 47 .Briefly, two different gRNA sequences were designed by using the "CRISPR Guides" tool available in the Benchling work environment (https:// benchling.com).Target sequences with the highest on-target score and the lowest off-target score were selected.Two gRNAs were designed to target the genes coding for the 4 copies of the MIB-MIP system in Mferi (locus_tags MF5583_00301 -MF5583_00308), by using complementary primers (#084-087).The corresponding pgRNA plasmids (pgRNA-1MIB-MIP, pgRNA-2MIBMIP) were constructed following the protocol described elsewhere 48 .Plasmids were sequence verified using Sanger sequencing (Microsynth) and purified using QIAprep Miniprep Spin Kit (Qiagen).Plasmids were then transformed into S. cerevisiae W303a-eSpCas9 via lithium acetate transformation 49 and transformants were selected on SD-Trp-Ura medium (Takara).Recombination templates containing the yeast elements (CEN-ARS + HIS3) and the tetracycline resistance cassette (pS'tetM) were produced by PCR amplification of the ARS4/CEN6/HIS/ pS'tetM loci from the plasmid pMT85-PSTetM-ARSCenHis-pRS313 20 using primers #082 and #083 with the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and purified using the High Pure PCR Product Purification Kit (Roche).The resulting recombination template contained sequences with 50 bp homology to the regions flanking the MIB-MIP locus of the Mferi genome.Mferi genomes were isolated and introduced in S. cerevisiae W303a-eSpCas9 pgRNA-1MIBMIP or pgRNA-2MIBMIP by spheroplast transformation 50 , as previously described 13 .Yeast Artificial Chromosomes (YACs) containing the modified Mferi genomes were isolated as previously described using the CHEF Mammalian Genomic DNA Plug kit (BioRad) 14 .

Genome transplantation into Mcap ΔRE recipient cell
The genome IVB14/OD_0535::YCp-ΔMIB-MIP maintained in S. cerevisiae as a YAC was transplanted into Mcap ΔRE strain as previously described 13,23 .Briefly, Mcap ΔRE was grown in SOB+ medium until early stationary phase (pH 6.5), washed in 10 mM Tris 250 mM NaCl pH 6.5 and resuspended in cold 0.1 M CaCl 2 .Transplantation was carried out mixing SP5 without serum and agarose plugs containing the modified genome with the resuspended cells in 2X Fusion Buffer (20 mM Tris, 20 mM MgCl 2 , 500 mM NaCl, 10% PEG 6000 pH 6.5).Mixtures were incubated statically for 90 min at 30 °C and then plated in SP5 plates supplemented with 15 µg mL −1 tetracycline.The resulting transplanted mutant strains were passaged three times in liquid selective medium and pre-screened by MALDI-TOF MS identification using a Microflex LT instrument (Brucker).Moreover, clones were also subjected to multiplex and simplex PCRs to confirm integrity of the genome in selected transplanted clones.Additionally, the genomes of mutant strains were verified by PacBio sequencing and mapping assembly to the Mferi IVB14/OD_0535 parental strain 15 .Full genome sequence can be found in BioProject PRJNA1062711.

Growth rate determination
Growth rate of the different mycoplasma strains was assessed by colorchanging units (CCU) per mL as well as colony-forming units (CFU) for up to 20 h every 60 min.Briefly, 100 mL SP5 at pH = 7.5 containing 10 2 cells mL −1 were incubated at 37 °C, and 5% CO 2 and small aliquots were removed every hour.Each aliquot was serially diluted and distributed in 200 µL volumes using 96-well plates (for CCU calculation) and plated as spot dilutions in SP5 agar plates (for CFU calculation).Color change and colonies were assessed after 2 days of incubation.To better compare the growth rate between wild-type (WT) and mutant strains, no additional antibiotics were added to the SP5 medium during the experiment.Growth curves were plotted, and the growth rates were calculated using GraphPad Prism v9.0.0

Plasmid construction
All plasmids generated in this study are listed in Supplementary Table S2.All plasmids were constructed using the NEBuilder HiFi DNA Assembly kit (New England Biolabs).The plasmid pIVB03 was constructed by replacing the origin of replication of Mmc GM12 present in pMYCO1 51 by the origin of replication of Mferi type strain G5847 T11 , amplified with primers #001 and #002.The plasmid pIVB04 is a derivate of pIVB03 in which the tetM marker under the control of the spiralin promoter (pS') was replaced by pS'pac marker, amplified from the gDNA of Mcap ΔRE strain 23 using primers #003 and #004.The plasmid pIVB06 is a derivate of pIVB04 in which the orientation of the pS'pac marker was switched.This switch was performed by amplifying the pIVB04 backbone without the pS'pac marker using primers #007 and #008 and the pS'pac marker using primers #005 and #006.Plasmid pIVB08 is a derivate of plasmid pIVB03 and was constructed by assembling the pS'tetM marker amplified with primers #011 and #012 with the pIVB03 backbone amplified in two parts using primers #009 with #010, and #013 with #014.The plasmid pIVB09 was built by replacing the pS'tetM cassette from pIVB08 by the pS'pac marker, employing primers #009 and #016 for the amplification of the pIVB08 backbone and primers #011 and #015 to amplify the pS'pac marker.
All plasmids carrying MIB-MIP copies of the different Mollicutes have the pIVB09 as a backbone, amplified using primers #017 and #018.The MIB-MIP gene pairs and their natural promoter regions were amplified from gDNA of each respective host strain (Mferi IVB14/OD_0535, M. hyopneumoniae Ue273, M. hyorhinis JF5820).To generate plasmids expressing MIB-MIP gene copies with the promoter region of the first MIB-MIP gene copy of Mferi IVB14/OD_0535 (P MM1mfe ), the pIVB09 backbone containing the P MM1mfe was amplified using primers #017 and #051 and each MIB-MIP copy with their respective primer set.Plasmid carrying the tagged MIB-MIP copy number 4 from Mmc was created amplifying the promoter sequence of the first MIB-MIP copy of Mmc using primers #057 and #058, and each gene with primers #059 and #060, and #061 and #062.Plasmids bearing codon-optimized MIB-MIP gene pairs tagged with 6xHis and FLAG, respectively, were created by cloning each gene copy, which was custom synthesized (GenScript).Optimization was carried out by using the Optimizer tool 52 , using the guided random method and the codon table of M. mycoides subsp.capri (species 40477).The plasmids carrying transcriptional fusions of the monomeric Kasubira-Orange2 (mKO2) gene with the promoter regions of MM2 mfe and MM3 mfe were built by assembling the mKO2 gene (custom synthesized by Genscript) amplified with primers #096/#097 and #098 with the promoter regions amplified with #021 + #094 and #023 + #095.The promoter-less version was created using primer #098 + #099.
All plasmids were sequence-verified by Sanger sequencing at Microsynth AG (Switzerland).Sequences of all plasmids can be found in the Supplementary Data 3.

Transformation of mycoplasmas and screening of mutants
The transformation protocol used to transform oriC-plasmids into Mferi IVB14/OD_0535 was adapted from the one used for transformation of Mmc 23 , with some modifications.Mferi was grown overnight (O/N) in SP5 with pH adjusted to 8.0 (SP5 pH8 ) until late logarithmic phase (~pH 7.0) to achieve the highest total CFU mL −1 (4 mL culture/ transformation).Cells were cooled on ice, pelleted at 4200 × g for 15 min at 4 °C and washed once in Sucrose/Tris Buffer (0.25 M Sucrose, 10 mM Tris-HCl, pH 7.0).Each cell pellet was resuspended in 400 µL cold CaCl 2 0.1 M and kept on ice for 30 min.In a 50 mL falcon tube, 10 µL of plasmid (600-1000 ng µL −1 ) were added to 400 µL Fusion Buffer 2X (0.5 M Sucrose, 20 mM Tris-HCl, 40% PEG8000, pH 7.0) and left at room temperature.After the incubation of 30 min on ice, the 400 µL cell suspension was added into the Falcon tube containing the Fusion Buffer 2X as well as the plasmid and mixed gently.The reaction was left incubating at 30 °C for 25-30 min.Then, fusion reaction was stopped by adding 9 mL of cold SP5 and inverting the tube once.Cells were recovered by centrifugation at 4200 × g for 15 min at 10 °C and the supernatant was carefully discarded.The cell pellet was resuspended in 1 mL fresh SP5 pH8 and incubated at 37 °C for 45-60 min before plating on selective agar plates.Colonies of transformants were visible after 48 h but were picked up on day 3-4 after transformation in 1.5 mL microcentrifuge tubes containing 1 mL SP5 pH8 with the appropriate antibiotic (i.e., 16 µg mL −1 puromycin or 15 µg mL −1 tetracycline).Transformants were passaged three times before screening.Correct transformants were verified by PCR of cell lysates obtained as previously described 20 .

Assessment of plasmid stability
Mferi strain carrying the pIVB09 plasmid was grown overnight in SP5 pH8 supplemented with 16 µg mL −1 of puromycin, serially diluted and plated in non-selective SP5 plates and SP5 plates containing 16 µg mL −1 of puromycin (Passage P 0 ).Stability and plasmid retention was studied along 10 passages (1:1000 dilution) in SP5 pH8 supplemented with two concentrations of puromycin (8 or 16 µg mL −1 ) or non-selective conditions.CFU mL −1 were counted for each passage in selective (16 µg mL −1 puromycin) and non-selective plates.Plasmid retention ratios were calculated by dividing CFU mL −1 obtained in selective plates by total CFU mL −1 in each passage.

MIB-MIP-derived IgG cleavage analysis
IgG cleavage assays were performed as described in refs.5,10, with some modifications.For all Mferi strains, cultures were grown O/N in SP5 pH8 with antibiotic selection, if necessary, until stationary phase.Next day, 1 mL fresh SP5 pH8 medium was inoculated with 50 µL of the O/N cultures maintaining the antibiotic selection when necessary and grown at 37 °C and 5% CO 2 until late logarithmic phase (~10 9 cells).Cells were spun down at 7000 × g for 10 min, washed once with SP5 w/o serum 20 , and spun down again in the same parameters.Pellets were resuspended in 35 µL of SP5 w/o serum containing 250 ng µL −1 of purified IgG from goat or swine serum (Sigma-Aldrich) and incubated at 37 °C for 45 min.Then, cells were pelleted at 7000 × g for 10 min, and supernatants were recovered and mixed with 7 µL 6× Laemmli buffer before boiling at 100 °C for 10 min.Supernatants were separated in a 10% SDS-PAGE and transferred to a PVDF membrane using a Trans-Blot Turbo Transfer System (BioRad).Membrane was blocked in PBS with 5% skimmed milk (Becton Dickinson) and 0.05% Tween-20 (Sigma).Antibodies and working dilutions are listed in Supplementary Table S3.Membrane was developed using SuperSignal West Pico PLUS Chemiluminiscent substrate (ThermoFisher Scientific).In the case of M. hyorhinis and M. hyopneumoniae, IgG cleavage determination was performed similarly, but cells were grown in Friis medium until early stationary phase and no subculture step was performed.Besides, incubation with IgG was extended to 2 h (M.hyorhinis) or 3 h (M.hyopneumoniae).

Analysis of expression of MIB, MIP and mKO2
Expression of C-terminal tagged MIB and MIP, or mKO2 was analyzed by immunoblot using anti-6xHis, anti-FLAG or anti-mKO2 antibodies, respectively (Supplementary Table S3). 1 mL of culture was used for each total protein extraction.Bacterial cells were washed twice in sterile PBS before suspended in 100 µL of PBS.Lysates were obtained by adding 20 µL 6× Laemmli buffer and boiling at 100 °C for 10 min.Lysates were separated in a 7.5% or 15% SDS-PAGE before blotting.The immunoblots were performed as described above.For membrane protein enrichment by Triton X-114 fractioning, samples were treated according to protocols published elsewhere 53,54 .

Fluorescence microscopy of Mferi colonies
Mferi cultures were diluted and seeded in puromycin selection plates and incubated at 37 °C 5% CO 2 for 2 days.Then, plates were observed under a Nikon Eclipse Ts2-FL inverted microscope with a TRITC filter cube and a DS-Fi3 camera under the control of NIS-Elements software.To take the fluorescence pictures, colonies were exposed for 800 ms.Composed images were merged by using ImageJ software.

Statistical analysis
All analyses were carried out using GraphPad Prism (v9.0.0).One-way ANOVA tests and Tukey's comparative tests were performed to assess significance and calculate p values when indicated.

In vivo infection of domestic goats
The experimental infection of goats was performed in compliance with the Swiss animal protection law (TSchG SR 455; TSchV SR 455.1;TVV SR 455.163) under the cantonal license BE67/19.The experiments were reviewed by the cantonal committee on animal experiments of the canton of Bern, (Switzerland) and approved by the cantonal veterinary authority (Amt für Landwirtschaft und Natur LANAT, Veterinärdienst VeD, Bern, Switzerland).We have complied with all relevant ethical regulations for animal use.
The infection trial was carried out at the Institute of Virology and Immunology (Mittelhäusern, Switzerland) using six female goats in total, aged between 1 and 4 years old and weighting 50-60 kg, and randomly split into two groups of three animals.Animals were infected intranasally with 1 mL 10 8 CCU mL −1 of Mferi strain G5847 T , or Mccp strain ILRI181 on two consecutive days as described recently 55 .One mL of mycoplasma culture was atomized using a 1 mL syringe attached with a MAD Nasal TM Intranasal Mucosal Atomization Device (Teleflex) and 500 µL of aerosol was applied to each nostril.At 4 days post-infection (dpi), each animal was infected transtracheally with the same number of color changing units (10 8 CCU mL −1 ).One mL of culture was followed by flushing with 5 mL sterile phosphate buffered saline.For the duration of the experiment, the animals were housed in a high containment facility, one stable per group of infection.The animals were kept on straw with a local ambient temperature of 20-22C . Before and after Mferi or Mccp infection, body temperature and clinical status was monitored daily.The clinical status was assessed by a veterinarian, always the same person to ensure unbiased clinical assessment.The rectal body temperature was measured with a digital thermometer, whereas respiratory and heart frequencies employed a stethoscope.Differential blood cell counts were determined from EDTA blood samples using an automated hematology analyzer (VetScan).Animals were euthanized when endpoint criteria were reached or at the end of the trial and subjected to postmortem analysis.Endpoint criteria were the same as reported elsewhere 55 .

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

MIB-MIP tandem gene copies of Mferi IVB14/OD_0535 are closely related to the ones present in Mmc
The MIB-MIP system is widespread between different members of the Mollicutes 5 and is a candidate virulence factor with potential implications in immune evasion 9,10 .Mferi strain G5847 T was shown to contain a large repeat of the MIB-MIP encoding region in the chromosome, leading to the presence of 2 identical sets of three MIB-MIP gene copies 13 .This unusual configuration is not present in any other isolate so far, which prompted us to study the occurrence and diversity of the MIB-MIP gene copies in Mferi IVB14/OD_0535 in respect to the rest of the Mollicutes.Therefore, we selected Mollicutes genomes, including the ones reported in this study, and searched for the presence of the MIB-MIP system.Altogether, we included a total of 34 genomes from 23 different species (Fig. 1A).We analyzed the sequences of 70 genes coding for putative MIB proteins and 71 genes encoding putative MIPs.In most genomes analyzed, both MIB and MIP counterparts were found adjacent in the same genetic locus, most likely forming a single transcriptomic unit.Occasionally we observed "orphan" MIB-or MIP-encoding genes.However, in these genomes another MIB-MIP pair was always present elsewhere in the genome, suggesting that Ig cleavage activity can potentially still occur.To facilitate the analysis, only the paired MIB-MIP proteins were included in the construction and analysis of the phylogenetic tree (Fig. 1A).
An initial scrutiny of the phylogenetic relations between MIB-MIP proteins of different species revealed three major distinctive branches, (i) encompassing Mycoplasma and Mycoplasmopsis species infecting ruminants; (ii) Ureaplasma, Metamycoplasma and some MIB-MIP pairs of porcine Mesomycoplasma species; and (iii) a more distant branch containing the MIB-MIP pairs of Mycoplasmoides and Mesomycoplasma species.We detected several examples of closely related MIB-MIP systems between Mollicutes species that are phylogenetically very distant but share the same hosts, such as Ureaplasma species and Metamycoplasma hominis, Mycoplasmoides gallisepticum and Mycoplasmopsis synoviae, or Mycoplasmopsis pulmonis and Metamycoplasma arthritidis, infecting humans, poultry, or rodents, respectively (Fig. 1A, B), as previously reported 5 .Here we found that there is a strong conservation between each pair in the same position, suggesting that multiple copies of this tandem system were present in a common ancestor of these mycoplasmas.Furthermore, there is a significant difference between the two strains of Mferi analyzed, as the MIB-MIP pairs of the type-strain G5847 T are similar to the ones present in Mccp, while the MIB-MIP pairs of the IVB14/OD_0535 strain cluster closely to the ones of the other members of the "M.mycoides cluster".Remarkably, the two paired MIB-MIP copies of M. hyopneumoniae, located on different chromosomal loci, are highly divergent, with one MIB-MIP being more similar to the only system present in M. hyorhinis and the other more similar to MIB-MIP present in Mesomycoplasma ovipneumoniae (Fig. 1A).Overall, our analysis shows that the MIB-MIP system of Mferi might vary significantly between isolates, suggesting horizontal gene acquisition from different ancestors to different members of this species.

Analysis of MIB-MIP expression in Mferi
We studied the expression of each MIB-MIP gene pair of this species by transcriptomics and proteomics analyses to see whether the gene pairs are organized as operons and to identify different promoters that can be used to express MIB-MIP pairs or other genes from other Mollicutes species.Exploration of the transcriptomic data showed that each MIB-MIP pair could constitute an individual transcriptional unit, with the first and last pairs (MM1 mfe and MM4 mfe ) being transcribed at higher levels than the other two pairs (MM2 mfe and MM3 mfe ) (Fig. 2A).Moreover, all MIB-MIP gene pairs are among the mid-to-high expressed genes of Mferi, with no big apparent differences between MIB and MIP transcription (Fig. 2B).However, when analyzing the proteomics data, MIB-MIP pairs are not among the highly expressed proteins of the cell highlighting the lack of correlation between RNA and protein levels (Fig. 2C).Besides, the protein expression levels of all MIP copies of each pair are significantly higher compared to their MIB counterparts.Given the similar disposition and number of the different MIB-MIP gene pairs between Mferi IVB14/OD_0535 and Mmc GM12, we also analyzed transcriptomics and proteomics data of GM12 obtained in a previous study 29 .Our results showed a similar mRNA expression trend for each MIB-MIP pair, with the first tandem of genes being transcribed higher than the rest (Supplementary Fig. S1).Moreover, proteomics data showed that most MIP proteins are expressed at higher levels than their respective MIB protein partner, with most of them being not reliably detected (Supplementary Fig. S1).Overall, these results suggest that each MIB-MIP system likely constitutes an individual expression unit and that expression of the different tandem of genes could be species-specific, despite having similar genetic organization or distribution in the different chromosomes.

Identification of MIB-MIP promoter and terminator regions
To further characterize the transcriptional profile shown by the RNAseq analysis of the MIB-MIP locus of Mferi, we decided to experimentally determine the transcriptional start sites (TSSs) present in that genomic area by 5' Rapid Amplification of cDNA Ends (5'RACE).We could detect a single band for all MIB-MIP gene tandems, except for the second MIB-MIP pair in which two discreet bands were obtained (Fig. 3A and Supplementary Fig. S2).In this case, the smaller band could correspond to a TSS derived from an active promoter, while the larger product starts immediately after a large inverted repeat element.With these results, we could pinpoint the Pribnow box of each pair and no clear −35 element, which is quite common in mycoplasma species 56 .Moreover, genetic analysis of the downstream regions of MIB-MIP gene clusters in a number of members of the "Mycoplasma mycoides cluster" revealed the presence of similar inverted repeat elements with a structure reminiscent of rho-independent terminators (Fig. 3B).These elements are 32-34 bp long and are located very close to the stop codon of the MIP genes.The last MIB-MIP gene pair is always devoid of this downstream element in all studied species, suggesting that it is part of a larger operon unit that also comprises putative ATPase-coding genes located downstream of the MIB-MIP cluster.The consensus sequence of these elements was determined as 5'TA(A/C)NATCCTTT(A/G)G-NT (A/T 2 )T(A/T 2 )-CTAAAGGATTTTT using all available sequences (Fig. 3C).Employing RNAfold 57 , the RNA structure of these downstream elements was predicted to be a small hairpin with a minimum free energy of approximately −12.70 kcal mol −1 (Fig. 3C).

Generation and characterization of a M. feriruminatoris ΔMIB-MIP strain
To examine the activity of the MIB-MIP systems of Mferi we decided to generate a knock-out mutant strain.Mferi strain IVB14/OD_0535 has several genes coding for a total of four MIB-MIP tandem systems clustered in a single chromosomal locus 15 , in a similar disposition as in Mmc GM12 5 (Fig. 4A).To assess activity of each unique MIB-MIP gene pair of Mferi we generated a MIB-MIP knock-out mutant (ΔMIB-MIP) by cloning and modifying the genome of Mferi in yeast prior transplantation of the modified genome to a new mycoplasma cell.The genome of Mferi IVB14/ OD_0535 was transformed into S. cerevisiae carrying the necessary plasmids to replace the chromosomal locus coding for the four MIB-MIP gene pairs (MF5583_00301 to MF5583_00308, ~20 Kb) by a recombination template using the CReasPy-Cloning method 47 .The recombination template was designed to replace the sequence encompassed from the starting codon of the first MIB copy until the stop codon of the last MIP copy, preserving any upstream or downstream non-coding regions.YACs containing the modified genome were transplanted into an Mcap ΔRE recipient cell to obtain the desired Mferi ΔMIB-MIP strain (Supplementary Fig. S3).This knock-out mutant could grow with a certain delay compared to the WT strain in the absence of antibiotics (Fig. 4B), with a doubling time of 50 ± 2 min compared to 45 ± 3 min of the WT in SP5 (Fig. 4C).This growth delay is similar to the one observed when the Mmc GM12 strain is compared to the same strain carrying the YCp1.1 element, suggesting that insertion of this genetic region might be impacting bacterial fitness, not the absence of MIB-MIP genes.

Development of replicative plasmids for M. feriruminatoris
The genetic tools currently available for manipulation of Mferi are limited and only the introduction and modification of the bacterial genome in yeast has been reported, which is technically challenging and time consuming.To accelerate the shuttle in of genes into Mferi, we decided to generate replicative plasmids based on the origin of replication (oriC) sequence of the chromosome (Fig. 5A), as previously done for other Mollicutes species 51,58-60 .We adapted the oriC-plasmid pMYCO1 for use in Mferi by exchanging the oriC region of Mmc by the oriC region of Mferi strain G5847.This first oriCplasmid, named pIVB03, could be successfully transformed in Mferi and promoted resistance to tetracycline at 15 µg mL −1 .However, as Mferi strains engineered in yeast already have a tetracycline resistance cassette, we developed a derivative plasmid of pIVB03 named pIVB04 carrying the puromycin acetyl transferase (pac) gene, which was reported to confer resistance to puromycin in other closely related Mollicutes species 61 .This puromycin resistance-conferring plasmid was transformed but did not produce puromycin-resistant colonies carrying pIVB04 (Fig. 5B).This result confirmed that the plasmid backbone could indeed replicate in Mferi, but that the resistance cassette in pIVB04 was probably not expressed correctly.This negative outcome was changed by switching the direction of the pac gene (Fig. 5A), which became plasmid pIVB06, and allowed the recovery of resistant colonies at 16 µg mL −1 puromycin.In other Mycoplasma species, oriC-plasmids have been shown to be more stable when reducing the length of the oriC region or by deleting the dnaA gene 21,58 .Therefore, we modified the oriC-plasmids pIVB03 and pIVB06 by replacing the dnaA gene of Mferi by the antibiotic resistance cassette, generating streamlined oriC-plasmids with resistance to tetracycline (pIVB08) or to puromycin (pIVB09) (Fig. 5A).Note that we also switched the orientation of the tetM cassette in plasmid pIVB08.These plasmid versions transformed with an efficiency up to four times higher than their parental plasmids with dnaA counterparts (Fig. 5B).As oriC-plasmids from Mollicutes are known to be rapididly lost under non-selective conditions, we tested the stability of the pIVB09 vector in the population of Mferi upon serial passaging.presence of the plasmid in the cells is heavily reduced in the absence of puromycin in the medium, but a small subpopulation of cells can retain it nevertheless (Supplementary Fig. S4).Thus, the constructed oriC-plasmids can be used for subsequent mutant phenotype complementation studies and as versatile backbones for heterologous expression systems reported in this study.

Expression of native MIB-MIP gene pairs of M. feriruminatoris in trans
Each unique MIB-MIP gene pair of Mferi was cloned in a pIVB09 backbone under the control of their own natural promoters previously determined by 5'RACE.The newly constructed Mferi ΔMIB-MIP strain was transformed with each of these plasmids individually.Positive clones were exposed to goat IgGs to assess cleavage activity (Fig. 5C).Despite analyzing multiple clones harboring each MIB-MIP gene pair,we could only detect IgG heavy chain cleavage in clones expressing the first and last gene tandems of the cluster (MM1 mfe and MM4 mfe ), while other clones expressing the other MIB-MIP pairs (MM2 mfe and MM3 mfe ) showed marginal cleavage or activity below the detection limit.The activity of the promoters driving the expression of MM2 mfe and MM3 mfe could be confirmed by using plasmids carrying transcriptional fusions to a fluorescent reporter (Supplementary Fig. S5).As the transcriptomics data suggested that the MM1 mfe and MM4 mfe gene tandems were expressed at higher levels than MM2 mfe and MM3 mfe , and that expression from the promoter region of MM1 mfe (P MM1mfe ) had no apparent interplay with any rho-independent terminator or upstream regulatory sequences, we decided to reintroduce the MM2 mfe and MM3 mfe gene pairs under the control of P MM1mfe in a ΔMIB-MIP genetic background.Under these conditions, all MIB-MIP gene pairs could be expressed, and mutants showed clear IgG cleavage activity (Fig. 5D), suggesting that all MIB-MIP gene pairs are functional in cellulo, and that the P MM1mfe and the P MM4mfe regions were sufficient to drive expression of two relatively large membrane-associated proteins organized as an operon in trans.In cellulo IgG cleavage by M. hyopneumoniae and M. hyorhinis Cleavage of immunoglobulins by the MIB-MIP system has been reported in Mollicutes of the formerly known "Spiroplasma phylogenetic group", i.e., Mmc 10 or Mferi 13 , but never in Mollicutes species like Mesomycoplasma spp.To determine if important porcine pathogens such as M. hyopneumoniae or M. hyorhinis can target and cleave host IgGs, we analyzed immunoglobulin cleavage activity of two strains isolated in Switzerland.Genome analysis revealed that M. hyopneumoniae Ue273 contains two complete MIB-MIP gene pairs and a single orphan MIB gene, while M. hyorhinis JF5820 only contains a single MIB-MIP gene pair 19 (Fig. 6A).This contrasts with many Mollicutes species of the "Spiroplasma phylogenetic group", where all MIB-MIP gene copies are clustered in a single chromosomal locus containing 3-4 complete MIB-MIP gene pairs.Interestingly, no ATPase gene cluster was found downstream of any of the different MIB-MIP copies in M. hyopneumoniae, as it is the case in most Mollicutes species.This ATPase gene cluster in M. hyopneumoniae is found in a different chromosomal location instead (Supplementary Fig. S6).Incubation with purified commercial IgGs isolated from naïve pig serum showed cleavage activity by both pathogens (Fig. 6B), with the heavy chain of the immunoglobulins being targeted.

Heterologous expression of MIB-MIP gene pairs of other Mollicutes
The availability of a ΔMIB-MIP strain together with a vector capable of expression of MIB-MIP pairs in trans in Mferi prompted us to complement this strain with MIB-MIP systems from other Mollicutes species.First, we cloned the last gene tandem of the MIB-MIP operon of Mmc GM12 (MM4 Mmc ) under the control of the promoter region of the first gene tandem of the same species (P MM1Mmc ), mimicking a similar disposition performed in situ at the chromosomal location in a previous work 10 .This construction was transformed in the ΔMIB-MIP strain and positive clones exhibit restored capacity to cleave goat IgGs (Fig. 6C, lane ΔM/M+pIVB09-MM4 Mmc ).Hereafter, we cloned the two complete MIB-MIP gene pairs and the single MIB-MIP gene tandem from M. hyopneumoniae Ue273 (MM1 Mhp and MM2 Mhp ) and M. hyorhinis JF5820 (MM Mhr ) in a pIVB09 backbone.In a first attempt, we introduced each MIB-MIP set under the control of their natural promoters, as previously done with the MIB-MIP genes of Mferi and Mmc.However, despite obtaining similar number of transformants carrying the different oriC-plasmids, no IgG cleavage was detected (Fig. 6C).To facilitate recombinant expression, we adapted all the MIB-MIP coding sequences to the codon usage of Mmc, the closest species of the "Mycoplasma mycoides cluster" to Mferi with an available characterized codon usage table (kazusa.or.jp) and replaced the natural promoters with the P MM1mfe , which proved capable of generating mRNA of similar length as previously shown in this study.However, transformants carrying these new constructs could neither cleave goat nor porcine IgGs (Fig. 6C), suggesting that the system was not active or could not be correctly exported, folded, or displayed at the membrane of Mferi cells.Interestingly, the wild-type strain of Mferi could bind and cleave porcine IgGs, despite the pig not being its natural animal host.
To further investigate this, we cloned in pIVB09 tagged-versions of the MIB-MIP systems of Mferi (MM4 Mfe ), Mmc (MM4 Mmc ), and the single MIB-MIP system of M. hyorhinis (MM Mhr ) to track protein expression by immunoblotting.All MIB genes were fused with a C-terminal 6xHis tag, while their MIP counterparts were tagged with a C-terminal FLAG tag.Analysis of Mferi ΔMIB-MIP strains carrying these plasmids showed that neither of the proteins forming the MIB-MIB system of M. hyrorhinis was expressed in these conditions (Fig. 7A), which explained the lack of IgG cleavage showed previously.Sequence analysis of the MIB-MIP systems of Mferi, Mmc, and M. hyorhinis showed significant differences in the N-terminal residues (Supplementary S7), which could prevent export of these proteins to the cell surface.To test this, we replaced the predicted signal peptides of the tagged MIB-MIP system of M. hyorhinis with the ones present in the MIB-MIP pair 1 of Mferi.Strains carrying this construction could correctly express the protease component of the MM Mhr , but not the binding protein (Fig. 7B).We also corroborated that gene complementation with a single functional partner (either MIB or MIP) cannot restore IgG cleavage capacity in the ΔMIB-MIP mutant (Supplementary Fig. S8).
Goats infected intranasally and transtracheally with Mycoplasma feriruminatoris did not develop disease Mferi is considered a promising candidate for the development of a vaccine chassis 13 .However, Mferi has only been isolated from wild caprinae 11 ; thus, data regarding its pathogenic potential in closely related domestic animals are absent.Therefore, we decided to assess the pathogenicity of the type-strain G5847 T of Mferi as the representative member of the species.We used a challenge model established for the phylogenetically related species Mmc 9 and modified for Mccp 55 , which is robust and reproducible 62 .Positive control was the highly virulent Mccp ILRI181 63 .Clinical evaluation was assessed daily and was carried out 10 days pre-infection up to 25 days postinfection (dpi).Goats infected with Mferi showed no clinical signs in contrast to animals infected with Mccp, which showed onset of clinical disease including elevated body temperature at 6-8 dpi (Fig. 8).This was followed by high fever (>40.5 °C for all animals), associated with respiratory distress, coughing, and wheezing (8-10 dpi), less movement and reduced intake of food.All criteria considered, this clinical evaluation led to a severity grade of 3 at 10 dpi; consequently, the three animals infected with Mccp were euthanized (Supplementary Fig. S8).All animals infected with either species did not show a clear difference in the hematological parameters compared to their baseline levels prior infection (Supplementary Fig. S9).Postmortem analysis did reveal contagious caprine pleuropneumonia (CCPP) typical pathomorphological changes including the detection of Mccp, while the animals infected with Mferi did not have any lesions pointing towards Mferi-related disease and Mferi could not be isolated from the animals.

Discussion
Mferi has so far only been isolated from wild ruminants such as Alpine ibex 11,12 .The ability to modify its genome using synthetic genomics tools 13 , the absence of the cell wall, its expected glycosylation capacity (as observed in other Mollicutes species 64 ), and its favorable growth attributes make it an appealing candidate for vaccine-and drug delivery especially in reference to the respiratory tract or cancer treatment 65 .
Surface expression of heterologous antigens in Mferi is desirable for future antigen presentation in a live vaccine chassis.We aimed to investigate heterologous surface expression and focused our work on the MIB-MIP system.To get an idea of the sequence conservation of the MIB-MIP system in our model strain, we analyzed the presence and genetic identity of the MIB-MIP system in our strain and compared it to other different species.It has been shown that several copies of such genes, homologous to the MIB-MIP system of Mmc, were frequently present in most species of Mycoplasma, Mycoplasmopsis, Metamycoplasma and Mesomycoplasma, and a token presence in Mycoplasmoides spp. 5 .Our Mferi strain IVB14/OD_0535 contains 4 copies of MIB-MIP pairs all clustered in the same genomic location, in a similar disposition as in Mmc GM12.Moreover, they are phylogenetically very closely related, in contrast with the MIB-MIP system present in Mferi type strain G5847 T .This fact suggests that these genes may have distinct origin between different isolates.Our transcriptomics and 5'RACE analyses show that each MIB-MIP pair may function as an individual transcriptional unit-an operon-with its own putative promoter and a short palindromic sequence resembling rho-independent terminator sequences.We showed that the promoter of the first and last gene tandems are significantly stronger than the other two, which may influence the transcription of downstream elements.The first promoter element may overcome the terminator element and drive the expression of other MIB-MIP pairs, as shown by the 5'RACE result of MIB2 (Fig. 3 and Supplementary Fig. 2).Despite the data presented here, it cannot be ruled out that other internal promoter regions exist and are used to transcribe the MIP-encoding genes, especially in the case of MIP3.As there is no putative rho-independent terminator after the coding region of MIP4,  the last promoter likely plays a role on the expression of the highly conserved putative ATP-synthase gene cluster situated immediately downstream.It is still not clear if MIB-MIP-related IgG cleavage requires the activity of the downstream ATPase, but our results show that the two gene clusters do not necessarily work in cis.In line with this, the MIB-MIP systems present in M. hyopneumoniae are unlinked to the ATPase gene cluster (Fig. 6A and Supplementary Fig. S6), which is still present but in a different genetic context in this species, and it has its own putative promoter.The putative promoter region of the ATPase gene cluster of M. hyopneumoniae might consist of a classic −10 region and relatively canonical −35 box, separated by a large series of Adenines.This configuration resembles others found in promoters with DNA slippage control in several Mollicutes 66 .Thus, it is debatable that independent promoter regions can drive the expression of these ATPase related genes, even in Mollicutes species where this cluster is located downstream of the MIB-MIP genes.Due to their reduced-size genomes, Mollicutes are thought to be devoid of many transcriptional regulatory elements, and rho-independent terminators have been suggested as major fine-tuning, transcription-controlling elements 67 , in conjunction with DNA supercoiling and RNA degradation 68 .
Here, we developed transformation protocols and oriC-type plasmid vectors to shuttle antigen-encoding genes into Mferi and to accelerate the testing of heterologous protein expression.Our growth curves of the wildtype strain of Mferi showed the rapid decline of viable cells after 20 h of cultivation in SP5 medium, coinciding with medium acidification below pH 7.This characteristic contrasts slightly with Mmc, a closely related, relatively fast-growing Mycoplasma species, which can survive and maintain high bacterial titers for longer time in acidic conditions.Loss of viability upon acidification in mycoplasmas is not exceptional 69 , thus the differences in low pH tolerance between Mferi and Mmc could be attributed to distinctive metabolic capabilities 11 or growth requirements in both species.This reduced tolerance towards lower pH prompted us to adapt the standard transformation protocols for mycoplasmas of the "M.mycoides cluster", which use cells harvested at pH 6.2-6.5 and PEG solutions buffered at a similar pH, for transformation of Mferi.Most transformable Mollicutes species have higher transformation efficiencies when harvested at late-log phase 70 .By increasing the initial pH of the SP5 medium from 7.5 to 8, we could significantly increase the bacterial titers of Mferi after an overnight growth to 1-3 × 10 9 CFU mL −1 at pH 7, right before cell titer decline, optimizing transformation efficiencies for this bacterium.Thus, we also adapted the pH of all transformation solutions to pH 7 to mimic the medium conditions at the harvesting point.When comparing growth of the Mferi WT strain with the MIB-MIP mutant strain we observed a small delay.However, this delay is also observed between the Mmc GM12 strain and the Mmc YCp1.1, in which no gene has been knocked out.This fact suggests that the presence of the YCp1.1 element in some mycoplasma genomes can have an impact on growth.It has been previously shown in other species that the presence of certain antibiotic markers influences mycoplasma growth even in the absence of the drug 71 , which might mask some mutant phenotypes.Thus, removal of antibiotic cassettes is highly advisable in future functional genomics studies in Mferi.
In this work, we also developed a series of replicative plasmids based on the modification of the origin of replication of the type-strain G5847 T that can be easily used in Mferi.Many species of Mollicutes can stably maintain episomal DNA containing the oriC sequence of the same species or a closely related one 21,51,[58][59][60][72][73][74] . For cetain species such as M. agalactiae, it has been found that the dnaA gene present in these oriC plasmids is not essential for replication and propagation.The removal of dnaA and simplification of the oriC region results in less frequent integration events at the chromosomal oriC locus in most species 21,58 , with the cost of usually lower transformation efficiency rates.On the contrary, in the case of oriC-plasmids derived from Mferi, removal of the dnaA gene resulted in 5 times higher transformation efficiencies regardless of the antibiotic marker used.Moreover, plasmids are rapidly lost in the absence of antibiotic pressure within the first passages (Supplementary Fig. S4), although it can stably remain in a small number of cells, suggesting spontaneous chromosomal integration.Only one oriC-plasmid developed in this work, pIVB04, in which the tetM marker had been replaced with pac marker did not yield any transformants despite having the same configuration as pIVB03 (Fig. 5).It was only when the orientation of the pac marker was flipped (plasmid pIVB06) that the plasmid yielded a similar number of transformants than pIVB03.This fact suggests that the oriC region contains promoter sequences that could challenge the transcription of the pS'pac cassette by antisense inhibition of gene expression.Antisense RNAmediated transcriptional attenuation in bacteria is well described [75][76][77] , involving either dsRNA-specific RNases, peptide nucleic acids, phosphorodiamidate morpholino oligomers or just by steric hindrance of transcription or translation.In Mollicutes, antisense RNAs have been identified in pathogenic species of swine such as M. hyopneumoniae 78 and human species like M. pneumoniae 79 or M. genitalium 80 , and their role in modulation of gene expression has been acknowledged.However, it seems likely that by steric obstruction, the RNA polymerase complex cannot successfully read through two genes with colliding orientations if both promoters are spatially close, which should result in a lower expression of both transcripts.This was shown in M. genitalium, when transposons expressing the toxic MG_428 gene coding for the alternative sigma factor σ 20 were all inserted in highly expressed genes in the opposite orientation of transcription, which dampened expression of the toxic gene and allowed cells to live 81,82 .In the case of pIVB04, most likely the promoter of the dnaN gene is interfering with the expression of the pac marker controlled by the spiralin promoter pS', interfering with the expression of the antibiotic cassette and limiting the puromycin resistance of transformed cells with the oriC-plasmid.This is not the case for pIVB03 and pMYCO1 oriC-plasmids, likely due to the larger size of the tetM marker (1.9 kb) compared to the pac cassette (0.6 kb).By using these oriC-plasmids, we could demonstrate that the promoter elements of the first MIB-MIP gene copy of Mferi (P MM1mfe ) was capable of successfully drive the expression of all MIB-MIP gene tandems individually in trans, which makes it a promising tool for recombinant expression of other rather large membrane proteins using this bacterium in the future.
Despite several attempts, expression of active heterologous MIB-MIP systems could only be achieved with a MIB-MIP system of Mmc, which is phylogenetically closely related to Mferi.Expression of functional MIB-MIP gene tandems from the porcine Mollicutes species M. hyopneumoniae and M. hyorhinis was not possible, despite the use of native promoters from Mferi or adapting the codon usage.The structure of the MIB-MIP tandem system has been recently obtained and characterized 10 and shows direct contacts between the two protein counterparts and with the targeted immunoglobulin.Therefore, correct export and folding of both proteins is likely pivotal for the system to work.We show that IgG cleavage of M. hyopneumoniae and M. hyorhinis in cellulo is possible under the standard laboratory conditions, which indicates that the systems present in these bacteria are active when expressed correctly.Despite our efforts, we failed at expressing active MIB-MIP systems from distantly related Mollicutes in Mferi, most likely due to problems related to the export of the system to the membrane.Despite many years of research, the protein export systems of Mollicutes are poorly characterized 83 .Most MIP proteins analyzed in this work contain a classic lipoprotein signal peptide (type II signal peptide), which consist of a positively charged N-terminal region, followed by a central hydrophobic area and a polar C-terminal region (lipobox) 84 .This signal peptide is also known to contain a preserved motif LXXC, which is recognized by the preprolipoprotein diacylglyceryl transferase (Lgt, encoded in MF5583_00077 and 00079 in Mferi IVB14/OD_0535) followed by the apolipoprotein N-acyltransferase (Lnt, encoded in MF5583_00341) that will create the linkage of the protein to the cell membrane after translocation via the Sec pathway 85 .However, none of the MIB proteins analyzed have a similar type II signal peptide or any clear transmembrane domain that suggests in silico association at the cell surface (Supplementary Fig. S10), aside from the interaction with MIP required for immunoglobulin cleavage.Furthermore, very low MIB protein levels are detected in our proteomics analyses in either Mmc or Mferi, as it was previously reported in Mmc 5 .This might be due to technical problems like low solubilization of MIB proteins during sample preparation or lack of suitable resulting peptides for MS analysis.However, in another closely related Mycoplasma species namely Mmm, also only the MIP proteins have been clearly detected in the surface proteome, while MIB proteins can only be seldomly detected 86 .Similarly, the closely related protein M, present in other Mollicutes species usually devoid of MIB-MIP systems 5 , also lacks any clear membrane anchoring signal and could not be identified in the protein membrane enriched fractions or cell surface protein labeling in a thorough proteomics study carried out in M. genitalium 87 .However, a recent study characterizing the protein M homolog (IbpM) from M. pneumoniae shows data indicating that this protein is located at the cell surface 88 , despite that advanced transmembrane domain predictors like DeepTMHMM 89 do not predict the presence of any transmembrane domain in neither protein M nor MIB.However, experimental data indicates that both MIB and MIP are associated to the membrane in Mferi (Supplementary Fig. S10) and also in M. bovis 8 .Understanding how these and other proteins lacking conventional signal peptides are exported in Mferi is crucial to develop functional display systems in this bacterium.
Finally, we investigated the pathogenicity of Mferi in domestic goats using an infection model that has been successfully used for phylogenetically closely related mycoplasmas.Mollicutes are reported to have high species tropism this should be confirmed for Mferi in an in vivo experiment.Our data do not point to pathogenicity in domestic goats and therefore this organism is unlikely to infect even phylogenetically more distant organisms, which is important for safety concerns.Only one infection route was tested, which is the main one in closely related bacteria of the "M.mycoides cluster" 90 .Goats challenged with Mccp, as expected, reached endpoint criteria at 10 dpi and were euthanized.Mferi could not be isolated from animals challenged with the latter, while Mccp was isolated from pathomorphological lesion typical of CCPP of the animals infected with it.The absence of Mferi from the post-mortem tissues investigated the fact that the animals cleared Mferi from the system.
In conclusion, we assessed pathogenicity of Mferi in an established animal model, developed new oriC-based vectors for rapid and versatile gene delivery in this microorganism, and use them to characterize expression of native and foreign anti-immunoglobulin systems of Mollicutes.This study provides new data regarding the molecular mechanisms of these specialized machineries that should aid in the understanding of the immune evasion strategies of pathogenic Mollicutes species.Moreover, we identified promoters suitable to drive expression of large heterologous surface proteins, which will be pivotal for future applications of Mferi as a bacterial vaccine chassis.

Fig. 1 |
Fig. 1 | Phylogenetic analysis of paired MIB-MIP systems in different Mollicutes species.A Unrooted phylogenetic tree representing concatenated MIB-MIP protein pairs in representative strains of different species of Mollicutes.Colors indicate the different phylogenetic groups: Mycoplasma (in dark red), Mycoplasmopsis (in green), Metamycoplasma (in dark yellow), Ureaplasma (in purple), Mesomycoplasma (in blue) and Mycoplasmoides (in turquoise).Animal hosts of the differentspecies are also displayed.The most distant branch is highlighted in gray.More information regarding strains and proteins displayed in the tree can be found in Supplementary TableS2.B Rooted phylogenetic tree showcasing the phylogenetic distance of representative strains of Mycoplasmatota according to the 16S RNA.Clostridium innocuum is displayed as an outgroup.Colors are used to distinguish between different phylogenetic groups as in (A).

Fig. 2 |
Fig. 2 | Analysis of MIB-MIP gene pair expression in Mferi using -omics.A Transcriptional analysis using RNAseq of three biological replicates.Top graph shows the mean fragments per kilobase of transcript per million mapped reads (FPKM) of each gene of the MIB-MIP cluster.Bottom graph indicates the read coverage obtained in each replicate.B Total gene distribution based on gene expression measured by RNAseq.C Total protein distribution based on protein expression measured by mass spectrometry.

Fig. 3 |
Fig. 3 | Analysis of the operonic structure of the MIB-MIP gene cluster.A Top section depicts the schematic organization of the chromosomal locus where the MIB-MIP gene cluster is located in M. feriruminatoris IVB14/OD_0535.Small arrows indicate promoter locations, while hairpins indicate the presence of putative terminator sequences.Bottom section illustrates the results obtained in the 5'RACE experiment.Arrows indicate the approximate location of the primers used in each gene.Length of each fragment obtained is also indicated.On the right, promoter sequences of each MIB copy are displayed.The transcriptional start site is indicated with a +1, and the corresponding Pribnow box is highlighted in bold letters and underlined.The starting codon of each gene is shown at the end.B Presence of large, inverted repeats after the MIP genes of several species of the "Mycoplasma mycoides cluster".First MIB-MIP operon of Mmm strain Gladysdale is interrupted by an insertion sequence.Mmm strain Gladysdale and M. leachii strain PG50 only have three MIB-MIP gene copies, therefore the last inverted repeat is absent.C Consensus sequence of the inverted repeat built with WebLogo tool.Sequence can form a hairpin loop with a minimum free energy (MFE) of −12.70 kcal/mol, as calculated with RNAfold webserver.

9 M 1 Fig. 4 |
Fig. 4 | Construction and characterization of a MIB-MIP deficient Mferi strain.A Schematic representation of the MIB-MIP operons and their genomic context in the ruminant mycoplasmas Mmc GM12 and Mferi IVB14/OD_0535.Deletion of the four MIB-MIP gene pairs of Mferi results in the ΔMIB-MIP strain, in which these genes (from the start codon of the first gene to the stop codon of the last gene, ~20 Kb) have been replaced by a tetracycline resistance cassette (PS'tetM) and yeast replication elements (YRE) (~4.1 Kb).B Growth curve determined by CFU mL −1 of at least three independent biological replicates of cultivated Mmc GM12 (in red), Mferi IVB14/OD_0535 (in blue), Mferi ΔMIB-MIP (in green), and Mmc GM12 YCp1.1 (in black).pH of the cultures of the wild-type strains at certain time-points is indicated.C Doubling time calculated during exponential phase of the four strains growing in standard SP5 medium at 37 °C without antibiotic selection.Results are the average of at least 3 biological replicates.*** p < 0.001, **** p < 0.0001.

Fig. 5 |
Fig. 5 | Construction of replicative oriC-plasmids for M. feriruminatoris.A Overview of the oriCplasmids designed in this work.B Transformation efficiency upon introduction of the different shuttle vectors in M. feriruminatoris.A total of four independent biological replicates were conducted.Transformants with pIVB03 and pIVB08 were selected with 15 µg mL −1 tetracycline, while 16 µg mL −1 puromycin was used to select transformants with pIVB04, pIVB06 and pIVB09.Data was analyzed using one-way ANOVA tests with Tukey's multiple comparisons test.** p < 0.005, *** p < 0.001, n.s.non-significant.C IgG cleavage activity of several strains of Mferi determined by Western blot.Cleaved IgG heavy chain is indicated with an arrow.Strain Mferi ΔMIB-MIP is abbreviated as ΔM-M in this panel and in (D).D IgG cleavage activity in a MIB-MIP mutant strain can be restored with MIB-MIP pairs 2 and 3 if they are under the control of the P MMmfe1 promoter, measured by Western blot.

Fig. 6 |
Fig. 6 | IgG cleavage activity in porcine Mesomycoplasma species.A Schematic representation of the chromosomal location of the MIB-MIP systems of M. hyopneumoniae and M. hyorhinis.B Porcine IgG cleavage activity of M. hyopneumoniae and M. hyorhinis analyzed by Western blot.C Porcine and goat IgG cleavage activity of Mferi ΔMIB-MIP strain expressing heterologous MIB-MIP systems.

Fig. 7 |
Fig. 7 | Heterologous MIB-MIP expression analysis in Mferi.A Schematic representation of three plasmids carrying tagged versions of MIB-MIP copies from different species (top).Detection of 6xHis and FLAG-tagged MIB and MIP, respectively.DnaK was used as a loading control (bottom).B Schematic representation of the two plasmids used to assess the role of the signal peptides in the expression of the MIB-MIP system of M. hyorhinis (top).Detection of 6xHis and FLAG-tagged MIB and MIP, respectively.DnaK was used as a loading control (bottom).

Fig. 8 |
Fig. 8 | Monitoring of body temperature during the animal challenge.Two groups of three outbreed goats were infected with either Mccp ILRI181 (in red) or Mferi G5847 T (in blue).Body temperature was monitored daily for both groups until experiment termination.Goats infected with Mccp were euthanized at day 10 due to the severity of their clinical signs, as indicated with a cross.